Project Summary Globally, more than 18 million people are infected with HIV, 10.4 million people are infected with tuberculosis, and over 200 million people become infected by malaria in a single year. There is a critical unmet need for vaccines against these infections and studies using mouse models have not yielded effective vaccines. The development of a functional memory response that enables the immune system to react quickly and robustly to previously experienced pathogens is the single most important factor for vaccine efficacy. Defining the mechanisms by which memory T cell responses are generated in humans is essential for developing efficacious vaccines. Previous studies in laboratory mice have shown that primary T cell responses to novel antigens originate from nave T cells. However, mice maintained under specific pathogen-free conditions are vastly different from humans in the extent of their microbial exposure. Our lab was the first to describe that human adults possess pre-existing memory phenotype (TMP) CD4+ T cells specific for viral antigens to which they have never been exposed. We further demonstrated that nearly a quarter of HIV-reactive T cell clones isolated from uninfected people also respond to unrelated microbial peptides, suggesting that these TMP cells are broadly cross-reactive to many different antigens. The development of TMPs likely reflects the cumulative exposure to environmental antigens following thymic development and these cells may be the earliest cells to respond during a primary infection. Here, we will build on these foundations and use yellow fever virus (YFV) vaccination as a model to test the hypothesis that pre-primed TMP cells generate potent effectors and preferentially give rise to post-vaccination memory T cells in humans. We will use a specific cell-labeling reagent called tetramers to tag T cells that recognize YFV antigens before vaccination and at multiple times after vaccination. This will allow us to trace YFV-reactive T cells directly ex vivo as they evolve following vaccination. Specifically, we will address the following questions: (1) does TMP cells have a greater functional diversity and plasticity compared to classic memory T cells; (2) does the abundance or the composition of TMP cells predict the rate and the magnitude of the effector response; (3) does TMP cells preferentially give rise to post-vaccination memory T cells? This study will reveal basic paradigms of how pre- existing memory precursors impact the potency and durability of CD4+ T cell immunity in vaccination. Understanding how the environment conditions the human T cell repertoire to influence later responses extends beyond vaccine efficacy and is broadly relevant to many areas of human health, including infection, autoimmunity, and cancer surveillance